Calcium Salted Spherical Nucleic Acids

This application is a U.S. National Phase of International Application No. PCT/US23/66675, filed May 5, 2023, which claims the priority benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63/338,736, filed May 5, 2022, which is incorporated herein by reference in its entirety.

This invention was made with government support under grant numbers 5U54CA199091-05, 5R01CA208783-05, and P50CA221747 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the text file containing the Sequence Listing is “2022-019_Seqlisting.XML”, which was created on Apr. 27, 2023 and is 49,675 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.

The disclosure is generally related to calcium salted spherical nucleic acids (SNAs). SNAs comprise a nanoparticle core surrounded by a shell of oligonucleotides. Methods of making and using the SNAs are also provided herein.

Development of therapeutic inhibitory oligonucleotides (e.g., siRNAs) has been limited because inhibitory oligonucleotides on their own cannot enter the target cells due to their poor stability in the blood stream and rapid clearance from circulation, rendering them impotent for systemic delivery.

Spherical nucleic acids (SNAs) provide distinct properties to overcome the challenges of using inhibitory oligonucleotides compared to their linear counterpart with enhanced cellular uptake and resistance to nuclease degradation. However, inhibitory oligonucleotide sequence dependent and cell-line differences can lead to decreased gene regulation efficiency of the inhibitory oligonucleotide functionalized SNA construct. In some aspects, the present disclosure provides CaCl) salted SNAs that significantly improve the gene regulation activity of the SNA by more than 20-fold independent of the inhibitory oligonucleotide sequence functionalized to the SNA surface. Improved gene regulation efficiency of the CaCl) salted inhibitory oligonucleotide-SNAs provide for the development and commercialization of SNAs that target a variety of genes involved in multiple disorders, including cancers and various genetic diseases.

Applications of the technology described herein include, but are not limited to:

Advantage of the technology described herein include, but are not limited to:

Accordingly, in some aspects the disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the external surface of the nanoparticle core, wherein each oligonucleotide in the shell of oligonucleotides comprises a phosphate backbone; the SNA comprising Caions adsorbed to the phosphate backbone of one or more oligonucleotides in the shell of oligonucleotides. In any of the aspects or embodiments of the disclosure, Caions are adsorbed to the phosphate backbone of each oligonucleotide in the shell of oligonucleotides. In further aspects, the disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the external surface of the nanoparticle core, wherein one or more oligonucleotides in the shell of oligonucleotides comprises a phosphate backbone; the SNA comprising Caions adsorbed to one or more oligonucleotides in the shell of oligonucleotides. In some embodiments, each oligonucleotide in the shell of oligonucleotides comprises a phosphate backbone. In further embodiments, Caions are adsorbed to the phosphate backbone of one or more oligonucleotides in the shell of oligonucleotides. In various embodiments, Caions are adsorbed to the phosphate backbone of each oligonucleotide in the shell of oligonucleotides. In some aspects or embodiments of the disclosure, Caions are adsorbed to one or more nucleobases of one or more oligonucleotides in the shell of oligonucleotides. In various embodiments, the SNA has a zeta potential that is about −40 millivolts (mV) to about −10 mV. In some embodiments, the SNA has a zeta potential that is about −10 mV. In various embodiments, the nanoparticle core is a metallic core, a semiconductor core, an insulator core, an upconverting core, a micellar core, a dendrimer core, a liposomal core, a lipid nanoparticle core, a polymer core, a metal-organic framework core, a protein core, or a combination thereof. In some embodiments, the polymer is polylactide, a polylactide-polyglycolide copolymer, a polycaprolactone, a polyacrylate, alginate, albumin, polypyrrole, polythiophene, polyaniline, polyethylenimine, poly(methyl methacrylate), poly(lactic-co-glycolic acid) (PLGA), or chitosan. In further embodiments, the nanoparticle core is gold, silver, platinum, aluminum, palladium, copper, cobalt, indium, cadmium selenide, iron oxide, fullerene, metal-organic framework, silica, zinc sulfide, or nickel. In some embodiments, the nanoparticle core is a liposome. In further embodiments, the liposome comprises a lipid selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), and cholesterol. In some embodiments, one or more oligonucleotides in the shell of oligonucleotides is attached to the external surface of the nanoparticle core through a lipid anchor group. In further embodiments, the lipid anchor group is attached to the 5′ end or the 3′ end of the one or more oligonucleotides. In further embodiments, the lipid anchor group is tocopherol or cholesterol. In some embodiments, one or more oligonucleotides in the shell of oligonucleotides is modified on its 5′ end and/or 3′ end with dibenzocyclooctyl (DBCO). In some embodiments, one or more oligonucleotides in the shell of oligonucleotides is modified on its 5′ end and/or 3′ end with a thiol. In further embodiments, the nanoparticle core and one or more or oligonucleotides in the shell of oligonucleotides comprise complementary reactive moieties that together form a covalent bond. In still further embodiments, the reactive moiety on the nanoparticle core comprises an azide, an alkyne, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an isothiocyanate, a N-hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, a quadricyclane, or a carbodiimide. In some embodiments, the reactive moiety on the one or more or each oligonucleotide in the shell of oligonucleotides is on a terminus of the oligonucleotide. In further embodiments, the reactive moiety on the one or more or each oligonucleotide in the shell of oligonucleotides comprises an alkyne, an azide, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an isothiocyanate, a N-hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, a quadricyclane, or a carbodiimide. In still further embodiments, the alkyne comprises dibenzocyclooctyl (DBCO) alkyne or a terminal alkyne. In some embodiments, the nanoparticle core comprises an azide reactive moiety and the one or more or oligonucleotides in the shell of oligonucleotides comprises an alkyne reactive moiety, or vice versa. In some embodiments, the alkyne reactive moiety comprises a DBCO alkyne. In some embodiments, the shell of oligonucleotides comprises about 5 to about 600 oligonucleotides. In further embodiments, the shell of oligonucleotides comprises about 5 to about 500 oligonucleotides. In still further embodiments, the shell of oligonucleotides comprises about 75 to about 100 oligonucleotides. In some embodiments, each oligonucleotide in the shell of oligonucleotides is about 5 to about 100 nucleotides in length. In some embodiments, each oligonucleotide in the shell of oligonucleotides is about 10 to about 50 nucleotides in length. In further embodiments, each oligonucleotide in the shell of oligonucleotides is about 20 to about 25 nucleotides in length. In some embodiments, each oligonucleotide in the shell of oligonucleotides has the same nucleotide sequence. In some embodiments, the shell of oligonucleotides comprises at least two oligonucleotides having different nucleotide sequences. In various embodiments, the shell of oligonucleotides is comprised of single-stranded DNA oligonucleotides, double-stranded DNA oligonucleotides, or a combination thereof. In further embodiments, the shell of oligonucleotides is comprised of single-stranded, double-stranded RNA oligonucleotides, or a combination thereof. In some embodiments, at least one oligonucleotide in the shell of oligonucleotides comprises a detectable marker. In some embodiments, the at least one oligonucleotide in the shell of oligonucleotides is configured to associate (e.g., hybridize) to a target analyte. In some embodiments, each oligonucleotide in the shell of oligonucleotides is configured to associate (e.g., hybridize) to a target analyte. In some embodiments, the detectable marker is attached to a polynucleotide hybridized to the at least one oligonucleotide in the shell of oligonucleotides. In some embodiments, the detectable marker attached to the polynucleotide is quenched when the polynucleotide with the detectable marker is hybridized to the at least one oligonucleotides in the shell of oligonucleotides. In some embodiments, the detectable marker attached to the polynucleotide is quenched when the polynucleotide with the detectable marker is hybridized to each oligonucleotide in the shell of oligonucleotides. In various embodiments, the shell of oligonucleotides comprises an inhibitory oligonucleotide, an immunostimulatory oligonucleotide, a targeting oligonucleotide, or a combination thereof. In further embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme. In some embodiments, the immunostimulatory oligonucleotide comprises a CpG nucleotide sequence. In further embodiments, the immunostimulatory oligonucleotide is a toll-like receptor (TLR) agonist. In still further embodiments, the TLR is chosen from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13). In some embodiments, the TLR is TLR9. In some embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO: 1). In some embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO: 3). In further embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCCATGACGTTCCTGACGTT (Spacer-18 (hexaethyleneglycol))Cholesterol-3′ (SEQ ID NO: 2). In still further embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCGTCGTTTTGTCGTTTTGTCGTT (Spacer-18 (hexaethyleneglycol))Cholesterol-3′ (SEQ ID NO: 4). In some embodiments, the SNA has an intercalating dye intensity that is decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to intercalating dye intensity of a NaCl-salted SNA under identical conditions.

In some aspects, the disclosure provides a composition comprising a plurality of the spherical nucleic acids (SNAs) of the disclosure. In some embodiments, the composition further comprises a therapeutic agent.

The disclosure also provides, in various aspects, a method of making a calcium chloride (CaCl)) salted spherical nucleic acid (SNA), the SNA comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the external surface of the nanoparticle core, wherein each oligonucleotide in the shell of oligonucleotides comprises a phosphate backbone; the method comprising: combining the nanoparticle core, a plurality of oligonucleotides, and calcium chloride (CaCl)) to create a mixture, wherein the combining results in the plurality of oligonucleotides becoming attached to the nanoparticle core to create the shell of oligonucleotides attached to the external surface of the nanoparticle core, thereby resulting in the CaCl) salted SNA. In some embodiments, the method further comprises isolating the CaCl) salted SNA from the mixture. Thus, in some aspects, the disclosure provides a method of making a calcium chloride (CaCl)) salted spherical nucleic acid (SNA), the SNA comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the external surface of the nanoparticle core, wherein each oligonucleotide in the shell of oligonucleotides comprises a phosphate backbone; the method comprising: combining the nanoparticle core, a plurality of oligonucleotides, and calcium chloride (CaCl)) to create a mixture, wherein the combining results in the plurality of oligonucleotides becoming attached to the nanoparticle core to create the shell of oligonucleotides attached to the external surface of the nanoparticle core, thereby resulting in the CaCl) salted SNA, and then optionally isolating the CaCl) salted SNA from the mixture. In some embodiments, Caions are adsorbed to the phosphate backbone of each oligonucleotide in the shell of oligonucleotides of the CaCl) salted SNA. In various embodiments, the CacIsalted SNA has a zeta potential that is about −40 millivolts (mV) to about −10 mV. In further embodiments, the CaCl) salted SNA has a zeta potential that is about −10 millivolts (mV). In some embodiments, concentration of CaCl) in the mixture is 70 millimolar (mM) to about 350 mM. In further embodiments, concentration of CaCl) in the mixture is about 230 millimolar (mM). In various embodiments, the nanoparticle core is a metallic core, a semiconductor core, an insulator core, an upconverting core, a micellar core, a dendrimer core, a liposomal core, a polymer core, a metal-organic framework core, a protein core, or a combination thereof. In some embodiments, the polymer is polylactide, a polylactide-polyglycolide copolymer, a polycaprolactone, a polyacrylate, alginate, albumin, polypyrrole, polythiophene, polyaniline, polyethylenimine, poly(methyl methacrylate), poly(lactic-co-glycolic acid) (PLGA), or chitosan. In further embodiments, the nanoparticle core is gold, silver, platinum, aluminum, palladium, copper, cobalt, indium, cadmium selenide, iron oxide, fullerene, metal-organic framework, silica, zinc sulfide, or nickel. In various embodiments, the shell of oligonucleotides comprises about 5 to about 600 oligonucleotides. In some embodiments, the shell of oligonucleotides comprises about 5 to about 500 oligonucleotides. In some embodiments, the shell of oligonucleotides comprises about 75 to about 100 oligonucleotides. In further embodiments, each oligonucleotide in the shell of oligonucleotides is about 5 to about 100 nucleotides in length. In various embodiments, each oligonucleotide in the shell of oligonucleotides is about 10 to about 50 nucleotides in length. In some embodiments, each oligonucleotide in the shell of oligonucleotides is about 25 nucleotides in length. In some embodiments, each oligonucleotide in the shell of oligonucleotides has the same nucleotide sequence. In some embodiments, the shell of oligonucleotides comprises at least two oligonucleotides having different nucleotide sequences. In various embodiments, the shell of oligonucleotides is comprised of single-stranded DNA oligonucleotides, double-stranded DNA oligonucleotides, or a combination thereof. In some embodiments, the shell of oligonucleotides is comprised of single-stranded, double-stranded RNA oligonucleotides, or a combination thereof. In further embodiments, the shell of oligonucleotides is comprised of single-stranded DNA oligonucleotides, double-stranded DNA oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA oligonucleotides, or a combination thereof. In further embodiments, at least one oligonucleotide in the shell of oligonucleotides comprises a detectable marker. In various embodiments, the shell of oligonucleotides comprises an inhibitory oligonucleotide, an immunostimulatory oligonucleotide, or a combination thereof. In further embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme. In some embodiments, the immunostimulatory oligonucleotide comprises a CpG nucleotide sequence. In further embodiments, the immunostimulatory oligonucleotide is a toll-like receptor (TLR) agonist. In still further embodiments, the TLR is chosen from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13). In some embodiments, the TLR is TLR9. In further embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO: 1). In further embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO: 3). In some embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCCATGACGTTCCTGACGTT (Spacer-18 (hexaethyleneglycol))Cholesterol-3′ (SEQ ID NO: 2). In some embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCGTCGTTTTGTCGTTTTGTCGTT (Spacer-18 (hexaethyleneglycol))Cholesterol-3′ (SEQ ID NO: 4). In various embodiments, the CaCl) salted SNA is an SNA as described herein.

In further aspects, the disclosure provides a composition comprising a plurality of the CaCl) salted spherical nucleic acids (SNAs) produced by a method of the disclosure. In some embodiments, the composition further comprises a therapeutic agent.

In some aspects, the disclosure provides a method of inhibiting expression of a gene product comprising the step of hybridizing a target polynucleotide encoding the gene product with a CaCl) salted spherical nucleic acid (SNA) or composition of the disclosure, wherein hybridizing between the target polynucleotide and one or more oligonucleotides in the shell of oligonucleotides occurs over a length of the target polynucleotide with a degree of complementarity sufficient to inhibit expression of the gene product. In some embodiments, expression of the gene product is inhibited in vivo. In some embodiments, expression of the gene product is inhibited in vitro. In various embodiments, expression of the gene product is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to inhibition of gene product expression of a spherical nucleic acid (SNA) comprising a nanoparticle core and a shell of oligonucleotides attached to the external surface of the nanoparticle core, wherein each oligonucleotide in the shell of oligonucleotides comprises a phosphate backbone, and wherein the SNA does not comprise Caions adsorbed to the phosphate backbone of one or more oligonucleotides in the shell of oligonucleotides. In some embodiments, expression of the gene product is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to inhibition of gene product expression of a sodium chloride (NaCl)-salted spherical nucleic acid (SNA) under identical conditions. In further embodiments, the hybridizing occurs intracellularly. In some embodiments, accumulation of a spherical nucleic acid (SNA) of the disclosure within an endosome is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or more compared to accumulation of NaCl-salted SNAs within the endosome.

In further aspects, the disclosure provides a method of inhibiting expression of a gene product comprising the step of contacting a cell comprising the gene product with a SNA of the disclosure, thereby inhibiting expression of the gene product. In some embodiments, expression of the gene product is inhibited in vivo. In some embodiments, expression of the gene product is inhibited in vitro. In various embodiments, expression of the gene product is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to inhibition of gene product expression in a cell contacted with a NaCl-salted spherical nucleic acid (SNA) under identical conditions. In some embodiments, expression of the gene product is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to inhibition of gene product expression in a cell not contacted with a spherical nucleic acid (SNA). In further embodiments, accumulation of a spherical nucleic acid (SNA) of the disclosure within an endosome is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or more compared to accumulation of NaCl-salted SNAs within the endosome.

In further aspects, the disclosure provides a method of treating a disorder comprising administering an effective amount of a CaCl) salted SNA or composition of the disclosure to a subject in need thereof, wherein the administering treats the disorder. In various embodiments, the disorder is cancer, an infectious disease, an autoimmune disease, or a combination thereof.

In some aspects, the disclosure provides a method for detecting a target analyte comprising the step of contacting the target analyte with a CaCl) salted SNA or composition of the disclosure, wherein one or more oligonucleotides in the shell of oligonucleotides comprises a detectable marker, wherein the contacting results in binding of the target analyte to the one or more oligonucleotides in the shell of oligonucleotides comprising the detectable marker, resulting in a detectable change and thereby detecting the target analyte. In some embodiments, the detectable marker is attached to a polynucleotide hybridized to the one or more oligonucleotides in the shell of oligonucleotides, wherein association of the one or more oligonucleotides in the shell of oligonucleotides with the target analyte releases the polynucleotide hybridized to the one or more oligonucleotides in the shell of oligonucleotides and the detectable marker is detectable after release. In further embodiments, the detectable marker attached to the polynucleotide is quenched when the polynucleotide with the detectable marker is hybridized to the one or more oligonucleotides in the shell of oligonucleotides. In still further embodiments, the detectable marker is detectable only when the one or more oligonucleotides in the shell of oligonucleotides is associated with the target analyte. In some embodiments, the detectable marker is quenched when the one or more oligonucleotides in the shell of oligonucleotides is not associated with the target analyte. In various embodiments, the detectable marker is situated at an internal location within the oligonucleotide. In some embodiments, the binding results in restriction of internal rotation of the detectable marker. In further embodiments, the detectable marker is thiazole orange (TO), quinoline blue, quinoline violet, thiazole red, a derivative thereof, or a cyanine derivative. In some embodiments, the detectable change is proportional to concentration of the target analyte. In various embodiments, the target analyte is a protein, an ion, a small molecule, a lipid, a carbohydrate, an oligosaccharide, a cell, an oligonucleotide, or a combination thereof. In further embodiments, the target analyte is RNA. In some embodiments, the target analyte is mRNA. In some embodiments, the target analyte is cytosolic mRNA.

In further aspects, the disclosure provides a method for up-regulating activity of a toll-like receptor (TLR), comprising contacting a cell having the toll-like receptor with a CaCl) salted SNA or composition of the disclosure, thereby up-regulating activity of the TLR. In some embodiments, the shell of oligonucleotides comprises one or more oligonucleotides that is a TLR agonist. In further embodiments, the toll-like receptor is toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12, toll-like receptor 13, or a combination thereof. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo.

In yet additional aspects, the disclosure provides a method for down-regulating activity of a toll-like receptor (TLR), comprising contacting a cell having the toll-like receptor with a CaCl) salted SNA or composition of the disclosure, thereby down-regulating activity of the TLR. In some embodiments, the shell of oligonucleotides comprises one or more oligonucleotides that is a TLR antagonist. In various embodiments, the toll-like receptor is toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12, toll-like receptor 13, or a combination thereof. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo.

Gene regulation therapy with inhibitor oligonucleotides (e.g., siRNAs) is a promising alternative to traditional small molecule or protein-based therapeutics.Inhibitory oligonucleotides can be designed to specifically degrade complementary target mRNA upon binding to the RNA-induced silencing complex (RISC).Thus, RNA interference (RNAi) is a promising technology in which inhibitory oligonucleotides (e.g., small interfering RNAs (siRNAs)) can be designed to silence any target gene, exhibiting potential for treating diseases that are regarded as “undruggable” by conventional medicines. While inhibitor oligonucleotides such as siRNAs show therapeutic potential, they do not have the ability to readily enter cells on their own in part due to their negatively charged phosphate backbone, and they are susceptible to rapid degradation by nucleases, making it a challenge to broadly use them in clinical settings.

Inhibitory oligonucleotide (e.g., siRNA) conjugated spherical nucleic acids (SNAs), where inhibitory oligonucleotides are densely packed and radially oriented around a nanoparticle core into a spherical architecture provide distinct properties with enhanced cellular uptake, resistance to nuclease degradation without eliciting non-specific immune response.The SNA architecture has unique structure-dependent properties that can be exploited to overcome the challenges associated with the delivery of inhibitory oligonucleotides such as linear siRNA or antisense DNA. The three-dimensional oligonucleotide shell bestows SNAs with the ability to enter cells via scavenger receptor A-mediated endocytosis,and the dense shell that defines the SNA minimizes nuclease degradation.When siRNA-based SNAs enter the cytosol, they associate with the RISC complex to mediate gene silencing; thus, SNAs are promising entities for gene regulation therapeutics and have been used in hundreds of cell based knockdown experiments (e.g., EGFP11,12, GAPDH13, LUC214-17, EGFR18-20, Bcl2L1221,22, BcL223,24, GM3S25, HER226-28, MGMT29, Survivin20, MFG-E830, PLK112, IL17RA31, TGFβ32, STAT333, TIMP134, TNF-α34 and PDL135) and multiple human clinical trials.However, some decreased gene regulation efficiency of siRNA functionalized SNAs has been observed due to sequence dependent and cell-line dependent differences in the cytosolic delivery of the siRNA-SNA construct to associate with the RNA-induced silencing complex (RISC) for gene regulation. To realize potent gene regulatory activity, SNAs must be delivered to the cytosol to access the RISC complex and target mRNA, as is the case with all gene silencing therapies.The uptake pathway for SNAs involves trafficking through the endosomal pathway with accumulation in the late endosome, while only a small portion of the SNAs escape to the cytosol where they can engage in gene silencing.The extent of escape and corresponding activity are highly dependent upon sequence, cell type, and perhaps even cell cycle, making the identification of a lead structure for a given target a difficult-to-predict process. To improve the potency and increase the generality of siRNA-SNA based gene regulation constructs, a method that improves the cytosolic delivery of SNAs more broadly needs to be established and would be highly beneficial for biological and medical applications involving SNAs.

Hence, to improve inhibitory oligonucleotide-SNA's gene regulation efficiency, the present disclosure provides calcium chloride (CaCl)) salted SNAs (e.g., poly-lactic-co-glycolic acid (PLGA) SNAs) where Caions are bound to the phosphate backbone of the oligonucleotide shell. As shown in the Examples herein, CaCl) salted PLGA SNAs did not show any particle aggregation and exhibited increased zeta potential compared to regular PLGA SNAs (not salted with CaCl)). When treated to cells, CaCl) salted PLGA SNAs exhibited significantly improved gene regulation efficiency compared to regular PLGA SNAs by almost 20-fold independent of the siRNA sequence functionalized to the SNA surface without eliciting any cellular toxicity.

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

The terms “polynucleotide” and “oligonucleotide” are interchangeable as used herein.

As used herein, the term “about,” when used to modify a particular value or range, generally means within 20 percent, e.g., within 10 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent of the stated value or range.

Unless otherwise stated, all ranges contemplated herein include both endpoints and all numbers between the endpoints. The use of “about” or “approximately” in connection with a range applies to both ends of the range. Thus, “about 20 to 30” is intended to cover “about 20 to about 30”, inclusive of at least the specified endpoints.

A “subject” is a vertebrate organism. The subject can be a non-human mammal (e.g., a mouse, a rat, or a non-human primate), or the subject can be a human subject.

The terms “administering”, “administer”, “administration”, and the like, as used herein, refer to any mode of transferring, delivering, introducing, or transporting a SNA to a subject in need of treatment with such an agent. Such modes include, but are not limited to, oral, topical, intravenous, intraarterial, intraperitoneal, intramuscular, intratumoral, intradermal, intranasal, and subcutaneous administration.

As used herein, “treating” and “treatment” refers to any reduction in the severity and/or onset of symptoms associated with a disease (e.g., cancer). Accordingly, “treating” and “treatment” includes therapeutic and prophylactic measures. One of ordinary skill in the art will appreciate that any degree of protection from, or amelioration of, the disease (e.g., cancer) is beneficial to a subject, such as a human patient. The quality of life of a patient is improved by reducing to any degree the severity of symptoms in a subject and/or delaying the appearance of symptoms.

As used herein, a “targeting oligonucleotide” is an oligonucleotide that directs a SNA to a particular tissue and/or to a particular cell type or it is an oligonucleotide that detects a target analyte. In some embodiments, a targeting oligonucleotide is an aptamer. Thus, in some embodiments, a SNA of the disclosure comprises an aptamer attached to the exterior of the nanoparticle core, wherein the aptamer is designed to bind one or more receptors on the surface of a certain cell type, or the aptamer is designed to detect a target analyte. Target analytes contemplated by the disclosure include without limitation a protein, an ion, a small molecule, a lipid, a carbohydrate, an oligosaccharide, a cell, an oligonucleotide, or a combination thereof.

As used herein, an “immunostimulatory oligonucleotide” is an oligonucleotide that can stimulate (e.g., induce or enhance) an immune response. Typical examples of immunostimulatory oligonucleotides are CpG-motif containing oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA oligonucleotides, and double-stranded DNA oligonucleotides. A “CpG-motif” is a cytosine-guanine dinucleotide sequence. In any of the aspects or embodiments of the disclosure, the immunostimulatory oligonucleotide is a toll-like receptor (TLR) agonist (e.g., a toll-like receptor 9 (TLR9) agonist). In various embodiments, about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% 80%, 90%, 95%, or 100% of oligonucleotides in the shell of oligonucleotides of a SNA as described herein are immunostimulatory oligonucleotides.

The term “inhibitory oligonucleotide” refers to an oligonucleotide that reduces the production or expression of proteins, such as by interfering with translating mRNA into proteins in a ribosome or that are sufficiently complementary to either a gene or an mRNA encoding one or more of targeted proteins, that specifically bind to (hybridize with) the one or more targeted genes or mRNA thereby reducing expression or biological activity of the target protein. Inhibitory oligonucleotides include, without limitation, isolated or synthetic short hairpin RNA (shRNA or DNA), an antisense oligonucleotide (e.g., antisense RNA or DNA, chimeric antisense DNA or RNA), miRNA and miRNA mimics, small interfering RNA (siRNA), DNA or RNA inhibitors of innate immune receptors, an aptamer, a DNAzyme, or an aptazyme.

An “effective amount” or a “sufficient amount” of a substance is that amount necessary to effect beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. In the context of administering a SNA of the disclosure, for example, an effective amount is an amount sufficient to inhibit gene expression. Efficacy can be shown in an experimental or clinical trial, for example, by comparing results achieved with a substance of interest compared to an experimental control.

All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The present disclosure provides calcium salted spherical nucleic acids (SNAs) and compositions comprising calcium salted SNAs. As described herein above, calcium chloride (CaCl)) salted SNAs of the disclosure comprise Caions that are bound to one or more oligonucleotides (e.g., the phosphate backbone and/or the nucleobase) of the oligonucleotide shell. Calcium (Ca) ions can not only adsorb (bind) to the phosphate backbone of an oligonucleotide, but can also bind to the nucleobases, N7 and O6 atoms on guanine (G), N7 atom on adenine (A), O2 atom on cytosine (C) and O4 atom on uracil (U) or thymine (T) (see, e.g., J. Phys. Chem. B 2022, 126, 43, 8646-8654, Acc. Chem. Res. 2010, 43, 7, 974-984, and Langmuir, (2020), 5979-5989, 36 (21)). Thus, possible binding sites for Caon an oligonucleotide include both negatively charged phosphate oxygens of the phosphate backbone and the nitrogens and/or oxygens on the nucleobases. The disclosure therefore contemplates that in various embodiments there a SNA of the disclosure comprises or consists of between 1 to 3 Caions per nucleotide (each phosphate backbone has one Cabinding site while the nucleobases have additional binding sites). The amount of Caions adsorbed to a SNA of the disclosure may also be expressed as a percentage of the total available Cabinding sites on a SNA that are occupied by a Caion. In various embodiments, about, at least about, or less than about 5%, about, at least about, or less than about 10%, about, at least about, or less than about 15%, about, at least about, or less than about 20%, about, at least about, or less than about 25%, about, at least about, or less than about 30%, about, at least about, or less than about 35%, about, at least about, or less than about 40%, about, at least about, or less than about 45%, about, at least about, or less than about 50%, about, at least about, or less than about 55%, about, at least about, or less than about 60%, about, at least about, or less than about 65%, about, at least about, or less than about 70%, about, at least about, or less than about 75%, about, at least about, or less than about 80%, about, at least about, or less than about 85%, about, at least about, or less than about 90%, about, at least about, or less than about 95%, or about or less than about 100% of the total available Cabinding sites on a SNA are occupied by a Caion. As taught herein (see, e.g., Example 2), to verify the association of Caions in a SNA of the disclosure (a CaCl) salted SNA), one can conduct an exclusion assay using an intercalating dye. Intercalating dyes that may be used include, but are not limited to, Picogreen™ (Thermo Fisher Scientific Inc., Waltham, MA), ethidium bromide, thiazole orange (TO), SYBR green, and LAMP Fluorescent Dye (New England Biolabs Inc., Ipswich, MA).

Electrostatic adsorption of Caions to the oligonucleotides in the shell of oligonucleotides of a SNA would physically screen the oligonucleotide shell, preventing the dye from intercalating within the oligonucleotide and consequently would lead to a decrease in intercalating dye fluorescence intensity. However, a divalent cation chelator (e.g., ethylenediaminetetraacetic acid (EDTA)) would chelate the Caions and enable the intercalation of the dye within the oligonucleotide and lead to an increase in fluorescence intensity. Thus, in various embodiments, a SNA of the disclosure has an intercalating dye intensity that is decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to intercalating dye intensity of a NaCl-salted SNA under identical conditions. SNAs comprise a nanoparticle core surrounded by a shell of oligonucleotides. In various embodiments, an oligonucleotide shell is formed when at least 10% of the available surface area of the exterior surface of a nanoparticle core includes an oligonucleotide. In further embodiments at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the available surface area of the exterior surface of the nanoparticle core includes an oligonucleotide. The oligonucleotides of the oligonucleotide shell may be oriented in a variety of directions. In some embodiments the oligonucleotides are oriented radially outwards. The oligonucleotide shell comprises one or more oligonucleotides attached to the external surface of the nanoparticle core. The spherical architecture of the polynucleotide shell confers unique advantages over traditional nucleic acid delivery methods, including entry into nearly all cells independent of transfection agents and resistance to nuclease degradation. Furthermore, SNAs can penetrate biological barriers, including the blood-brain (see, e.g., U.S. Patent Application Publication No. 2015/0031745, incorporated by reference herein in its entirety) and blood-tumor barriers as well as the epidermis (see, e.g., U.S. Patent Application Publication No. 2010/0233270, incorporated by reference herein in its entirety).

Thus, in any of the aspects or embodiments of the disclosure, a spherical nucleic acid (SNA) is provided comprising (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the external surface of the nanoparticle core, wherein one or more oligonucleotides in the shell of oligonucleotides comprises a phosphate backbone; the SNA comprising Caions adsorbed to the phosphate backbone of one or more oligonucleotides in the shell of oligonucleotides. In some embodiments, each oligonucleotide in the shell of oligonucleotides comprises a phosphate backbone. In some embodiments, Caions are adsorbed to the phosphate backbone of each oligonucleotide in the shell of oligonucleotides. In further aspects, the disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the external surface of the nanoparticle core, wherein one or more oligonucleotides in the shell of oligonucleotides comprises a phosphate backbone; the SNA comprising Caions adsorbed to one or more oligonucleotides in the shell of oligonucleotides. In some embodiments, each oligonucleotide in the shell of oligonucleotides comprises a phosphate backbone. In further embodiments, Caions are adsorbed to the phosphate backbone of one or more oligonucleotides in the shell of oligonucleotides. In various embodiments, Caions are adsorbed to the phosphate backbone of each oligonucleotide in the shell of oligonucleotides. In any of the aspects or embodiments of the disclosure, Caions are adsorbed to one or more bases of one or more oligonucleotides in the shell of oligonucleotides. In some embodiments, the SNA has a zeta potential that is about −40 millivolts (mV) to about −10 mV, or about −20 mV to about −10 mV, or about −15 mV to about −10 mV. In further embodiments, the SNA has a zeta potential that is about −30 mV, −20, mV, −15 mV, or about −10 mV.

SNAs can range in size from about 1 nanometer (nm) to about 1000 nm, about 1 nm to about 900 nm, about 1 nm to about 800 nm, about 1 nm to about 700 nm, about 1 nm to about 600 nm, about 1 nm to about 500 nm, about 1 nm to about 400 nm, about 1 nm to about 300 nm, about 1 nm to about 200 nm, about 1 nm to about 150 nm, about 1 nm to about 100 nm, about 1 nm to about 90 nm, about 1 nm to about 80 nm in diameter, about 1 nm to about 70 nm in diameter, about 1 nm to about 60 nm in diameter, about 1 nm to about 50 nm in diameter, about 1 nm to about 40 nm in diameter, about 1 nm to about 30 nm in diameter, about 1 nm to about 20 nm in diameter, about 1 nm to about 10 nm, about 10 nm to about 150 nm in diameter, about 10 nm to about 140 nm in diameter, about 10 nm to about 130 nm in diameter, about 10 nm to about 120 nm in diameter, about 10 nm to about 110 nm in diameter, about 10 nm to about 100 nm in diameter, about 10 nm to about 90 nm in diameter, about 10 nm to about 80 nm in diameter, about 10 nm to about 70 nm in diameter, about 10 nm to about 60 nm in diameter, about 10 nm to about 50 nm in diameter, about 10 nm to about 40 nm in diameter, about 10 nm to about 30 nm in diameter, or about 10 nm to about 20 nm in diameter. In further embodiments, the SNA is, is at least, or is less than about 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20, or 10 nm in diameter (or in mean diameter when there are a plurality of SNAs). In further aspects, the disclosure provides a plurality of SNAs, each SNA comprising an oligonucleotide shell attached to the external surface of the nanoparticle core, wherein each oligonucleotide in the shell of oligonucleotides comprises a phosphate backbone; the SNA comprising Caions adsorbed to the phosphate backbone of one or more oligonucleotides in the shell of oligonucleotides. Thus, in some embodiments, the size of the plurality of SNAs is from about 10 nm to about 1000 nm (mean diameter), about 10 to about 900 nm in mean diameter, about 10 to about 800 nm in mean diameter, about 10 to about 700 nm in mean diameter, about 10 to about 600 nm in mean diameter, about 10 to about 500 nm in mean diameter, about 10 to about 400 nm in mean diameter, about 10 to about 300 nm in mean diameter, about 10 to about 200 nm in mean diameter, about 10 to about 150 nm (mean diameter), about 10 nm to about 140 nm in mean diameter, about 10 nm to about 130 nm in mean diameter, about 10 nm to about 120 nm in mean diameter, about 10 nm to about 110 nm in mean diameter, about 10 nm to about 100 nm in mean diameter, about 10 nm to about 90 nm in mean diameter, about 10 nm to about 80 nm in mean diameter, about 10 nm to about 70 nm in mean diameter, about 10 nm to about 60 nm in mean diameter, about 10 nm to about 50 nm in mean diameter, about 10 nm to about 40 nm in mean diameter, about 10 nm to about 30 nm in mean diameter, or about 10 nm to about 20 nm in mean diameter. In some embodiments, the diameter (or mean diameter for a plurality of SNAs) of the SNAs is from about 10 nm to about 150 nm, from about 30 to about 100 nm, or from about 40 to about 80 nm. In some embodiments, the size of the nanoparticles used in a method varies as required by their particular use or application. The variation of size is advantageously used to optimize certain physical characteristics of the SNAs, for example, the amount of surface area to which oligonucleotides may be attached as described herein. It will be understood that the foregoing diameters of SNAs can apply to the diameter of the nanoparticle core itself or to the diameter of the nanoparticle core and the oligonucleotide shell attached thereto. Further description of nanoparticle cores is provided herein below.

In various embodiments, the nanoparticle core is a metallic core, a semiconductor core, an insulator core, an upconverting core, a micellar core, a dendrimer core, a liposomal core, a lipid nanoparticle core, a polymer core, a metal-organic framework core, a protein core, or a combination thereof. In various embodiments, the polymer is polylactide, a polylactide-polyglycolide copolymer, a polycaprolactone, a polyacrylate, alginate, albumin, polypyrrole, polythiophene, polyaniline, polyethylenimine, poly(methyl methacrylate), poly(lactic-co-glycolic acid) (PLGA), or chitosan.

PLGA-SNAs may be synthesized using several strategies. In some embodiments, a PLGA SNA is synthesized by conjugating lipid-modified oligonucleotides to the surface of PLGA nanoparticles via hydrophobic-hydrophobic interactions. In some embodiments, a PLGA SNA is synthesized by conjugating oligonucleotide and the PLGA, which comprise complementary reactive moieties that together form a covalent bond. In some embodiments, DBCO-modified DNA strands are covalently conjugated to, e.g., azide groups through Cu-free click chemistry [Baskin, et al. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (43), 16793-16797]. While DBCO-modified oligonucleotides were used in examples herein, other alkyne moieties can be used instead, including a terminal alkyne (HC≡C—) or an internal alkyne (RC≡C—, where R comprises an alkyl). The alkyne moiety can also be attached to the oligonucleotide via a linker. In some embodiments, the reactive moiety on the nanoparticle core (e.g., a polymer comprising PLGA or in some embodiments PLGA-PEG) comprises an azide, an alkyne, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an isothiocyanate, a N-hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, a quadricyclane, or a carbodiimide. In further embodiments, the reactive moiety on the oligonucleotide is on a terminus of the oligonucleotide. In still further embodiments, the reactive moiety on the oligonucleotide comprises an alkyne, an azide, a maleimide, a thiol, an alcohol, an amine, a carboxylic acid, an olefin, an isothiocyanate, a N-hydroxysuccinimide, a phosphine, a nitrone, a norbornene, an oxanorbornene, a transcycloctene, an s-tetrazene, an isocyanide, a tetrazole, a nitrile oxide, a quadricyclane, or a carbodiimide. In some embodiments, the alkyne comprises dibenzocyclooctyl (DBCO) alkyne or a terminal alkyne. In further embodiments, the polymer (e.g., PLGA or PLGA-PEG) comprises an azide reactive moiety and the oligonucleotide comprises an alkyne reactive moiety, or vice versa. In still further embodiments, the alkyne reactive moiety comprises a DBCO alkyne. The PLGA-SNAs of the disclosure may contain a polymer selected from the group consisting of diblock poly(lactic) acid-poly(ethylene)glycol (PLA-PEG) copolymer, diblock poly(lactic acid-co-glycolic acid)-poly(ethylene)glycol (PLGA-PEG) copolymer, and combinations thereof. PLGA-SNAs are further described herein below and in International Publication No. WO 2018/175445, which is incorporated by reference herein in its entirety.

In some embodiments, the nanoparticle core is gold, silver, platinum, aluminum, palladium, copper, cobalt, indium, cadmium selenide, iron oxide, fullerene, metal-organic framework, silica, zinc sulfide, or nickel. In some embodiments, the nanoparticle core is a liposome. In further embodiments, the liposome comprises a lipid selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), and cholesterol.

Liposomes are spherical, self-closed structures in a varying size range comprising one or several hydrophobic lipid bilayers with a hydrophilic core. The diameter of these lipid based carriers range from 0.15-1 micrometers, which is significantly higher than an effective therapeutic range of 20-100 nanometers. Liposomes termed small unilamellar vesicles (SUVs), can be synthesized in the 20-50 nanometer size range, but encounter challenges such as instability and aggregation leading to inter-particle fusion. This inter-particle fusion limits the use of SUVs in therapeutics. In some aspects, liposomal spherical nucleic acids (LSNAs) comprise a liposomal core, a shell of oligonucleotides attached to the external surface of the liposomal core, the shell of oligonucleotides comprising one or more oligonucleotides comprising a phosphate backbone; the SNA comprising Caions adsorbed to the phosphate backbone of one or more oligonucleotides in the shell of oligonucleotides.

In various embodiments, one or more oligonucleotides in the shell of oligonucleotides is attached to the external surface of the nanoparticle core (e.g., liposomal core) through a lipid anchor group. In some embodiments, each oligonucleotide in the shell of oligonucleotides is attached to the external surface of the nanoparticle core through a lipid anchor group. In further embodiments, the lipid anchor group is attached to the 5′ end or the 3′ end of the at least one oligonucleotide. In still further embodiments, the lipid anchor group is tocopherol or cholesterol. Thus, in various embodiments, at least one of the oligonucleotides in the shell of oligonucleotides is an oligonucleotide-lipid conjugate containing a lipid anchor group, wherein said lipid anchor group is adsorbed into the lipid bilayer. In some embodiments, all of the oligonucleotides in the shell of oligonucleotides is an oligonucleotide-lipid conjugate containing a lipid anchor group, wherein said lipid anchor group is adsorbed into the lipid bilayer. In various embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the oligonucleotides in the shell of oligonucleotides is attached (e.g., adsorbed) to the exterior of the liposomal core through a lipid anchor group. The lipid anchor group comprises, in various embodiments, tocopherol, palmitoyl, dipalmitoyl, stearyl, distearyl, or cholesterol. While not meant to be limiting, any chemistry known to one of skill in the art can be used to attach the lipid anchor to the oligonucleotide, including amide linking or click chemistry.

Methods of making a liposomal SNA (LSNA) are described herein and are also described in, e.g., U.S. Pat. No. 10,182,988, incorporated by reference herein in its entirety.

Lipid nanoparticle spherical nucleic acids (LNP-SNAs) are comprised of a lipid nanoparticle core decorated with a shell of oligonucleotides. The lipid nanoparticle core comprises an ionizable lipid, a phospholipid, a sterol, and a lipid-polyethylene glycol (lipid-PEG) conjugate. The shell of oligonucleotides is attached to the external surface of the lipid nanoparticle core. The spherical architecture of the oligonucleotide shell confers unique advantages over traditional nucleic acid delivery methods, including entry into nearly all cells independent of transfection agents, resistance to nuclease degradation, sequence-based function, targeting, and diagnostics.

In some embodiments, the ionizable lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), C12-200, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), similar lipid/lipidoid structures, or a combination thereof. In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dihexadecanoyl phosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), monophosphoryl Lipid A (MPLA), or a combination thereof. In further embodiments, the sterol is 3β-Hydroxycholest-5-ene (Cholesterol), 9,10-Secocholesta-5,7,10 (19)-trien-3ß-ol (Vitamin D3), 9,10-Secoergosta-5,7,10(19),22-tetraen-3β-ol (Vitamin D2), Calcipotriol, 24-Ethyl-5,22-cholestadien-3ß-ol (Stigmasterol), 22,23-Dihydrostigmasterol (β-Sitosterol), 3,28-Dihydroxy-lupeol (Betulin), Lupeol, Ursolic acid, Oleanolic acid, 24α-Methylcholesterol (Campesterol), 24-Ethylcholesta-5,24(28)E-dien-3β-ol (Fucosterol), 24-Methylcholesta-5,22-dien-3β-ol (Brassicasterol), 24-Methylcholesta-5,7,22-trien-3β-ol (Ergosterol), 9,11-Dehydroergosterol, Daucosterol, or any of the foregoing sterols modified with one or more amino acids. In some embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate comprises 2000 Dalton (Da) polyethylene glycol. In further embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate is lipid-PEG-maleimide. In still further embodiments, the lipid-PEG-maleimide is 1,2-dipalmitoryl-sn-glycero-3-phosphoethanolamine (DPPE) conjugated to 2000 Da polyethylene glycol maleimide, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) conjugated to 2000 Da polyethylene glycol maleimide, or a combination thereof.

In any of the aspects or embodiments of the disclosure an oligonucleotide is attached to the external surface of a lipid nanoparticle core via a covalent attachment of the oligonucleotide to a lipid-polyethylene glycol (lipid-PEG) conjugate. In various embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the oligonucleotides in the shell of oligonucleotides are covalently attached to the exterior of the lipid nanoparticle core through the lipid-PEG conjugate. In various embodiments, one or more oligonucleotides in the oligonucleotide shell is attached (e.g., adsorbed) to the exterior of the lipid nanoparticle core through a lipid anchor group as described herein. In various embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the oligonucleotides in the shell of oligonucleotides is attached (e.g., adsorbed) to the exterior of the lipid nanoparticle core through a lipid anchor group as described herein. The lipid anchor group is, in various embodiments, attached to the 5′- or 3′-end of the oligonucleotide. In various embodiments, the lipid anchor group is tocopherol, palmitoyl, dipalmitoyl, stearyl, distearyl, or cholesterol.

The disclosure also provides methods of making calcium salted SNAs. Accordingly, in some aspects the disclosure provides a method of making a calcium chloride (CaCl)) salted spherical nucleic acid (SNA), the SNA comprising: (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the external surface of the nanoparticle core, wherein one or more or each oligonucleotide in the shell of oligonucleotides comprises a phosphate backbone; the method comprising: combining the nanoparticle core, a plurality of oligonucleotides, and calcium chloride (CaCl)) to create a mixture, wherein the combining results in the plurality of oligonucleotides becoming attached to the nanoparticle core to create the shell of oligonucleotides attached to the external surface of the nanoparticle core, thereby resulting in the CaCl) salted SNA, and then optionally isolating the CaCl) salted SNA from the mixture. In some embodiments, Caions are adsorbed to the phosphate backbone of one or more or each oligonucleotide in the shell of oligonucleotides of the CaCl) salted SNA. In some aspects or embodiments of the disclosure, Caions are adsorbed to one or more bases of one or more oligonucleotides in the shell of oligonucleotides. By way of example, following the combining of the nanoparticle core, the plurality of oligonucleotides, and calcium chloride (CaCl)) to create a mixture, the mixture is incubated to allow the plurality of oligonucleotides to become attached to the external surface of the nanoparticle core. The incubating may be performed at room temperature for about 6-24 hours, and may include shaking. In various embodiments, the mixture comprises a nanoparticle core (e.g., PLGA), a plurality of oligonucleotides, a surfactant (e.g., Poloxamer 188), a salt (e.g., NaCl), and CaCl). Various concentrations of CaCl) may be utilized in the mixture. In various embodiments, the concentration of CaCl) in the mixture is, is about, is at least about, or is less than about 7 mM, 36 mM, 50 mM, 70 mM, 100 mM, 130 mM, 150 mM, 160 mM, 184 mM, 200 mM, 210 mM, 230 mM, 290 mM, 333 mM, or 350 mM CaCl). In some embodiments, the concentration of CaCl) in the mixture is about 70 mM to about 350 mM. In further embodiments, the concentration of CaCl) in the mixture is about 230 mM.

In some embodiments, the calcium salted SNA is produced using a nanoprecipitation method. The plurality of oligonucleotides may be attached to the nanoparticle core using any method(s) understood in the art and/or described herein. For example and without limitation, the oligonucleotides may be attached to the nanoparticle core via copper-free click chemistry. In some embodiments, the oligonucleotides comprise a lipid anchor group such that they can adsorb to the external surface of the nanoparticle core (e.g., a liposomal core). The oligonucleotides and the nanoparticle core may also comprise complementary reactive moieties that together form a covalent bond. The resulting calcium salted SNAs may be isolated by any method known in the art, for example and without limitation, spin filtration. General methods of making SNAs are also described herein above.

In various embodiments, calcium salted SNAs have a zeta potential that is about-40 millivolts (mV) to about −10 mV. In some embodiments, the CaCl) salted SNA has a zeta potential that is about −10 millivolts (mV). In further embodiments, the CaCl) salted SNA has a zeta potential that is, is about, is at least about, or is less than about −40 mV, −30 mV, −20 mV, −10 mV, or −5 mV. Zeta potential is measured, for example and without limitation, using a Zetasizer (e.g., Malvern Zetasizer Ultra Red).

The disclosure provides spherical nucleic acids (SNAs) comprising (a) a nanoparticle core; and (b) a shell of oligonucleotides attached to the external surface of the nanoparticle core, wherein one or more or each oligonucleotide in the shell of oligonucleotides comprises a phosphate backbone; the SNA comprising Caions adsorbed to the phosphate backbone of one or more oligonucleotides in the shell of oligonucleotides. In various embodiments, the shell of oligonucleotides comprises an inhibitory oligonucleotide, an immunostimulatory oligonucleotide, a targeting oligonucleotide, or a combination thereof. Oligonucleotides contemplated for use according to the disclosure include those attached to a nanoparticle core through any means (e.g., covalent or non-covalent attachment). Oligonucleotides of the disclosure include, in various embodiments, DNA oligonucleotides, RNA oligonucleotides, modified forms thereof, or a combination thereof. In any aspects or embodiments described herein, an oligonucleotide is single-stranded, double-stranded, or partially double-stranded. In any aspects or embodiments of the disclosure, an oligonucleotide comprises a detectable marker. In various embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme. In various embodiments, the immunostimulatory oligonucleotide comprises a CpG nucleotide sequence. In some embodiments, the immunostimulatory oligonucleotide is a toll-like receptor (TLR) agonist. In further embodiments, the TLR is chosen from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13). In some embodiments, the TLR is TLR9. In some embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO: 1). In further embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO: 3). In still further embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCCATGACGTTCCTGACGTT (Spacer-18 (hexaethyleneglycol))Cholesterol-3′ (SEQ ID NO: 2). In still further embodiments, one or more oligonucleotides in the shell of oligonucleotides comprises or consists of the sequence of 5′-TCGTCGTTTTGTCGTTTTGTCGTT (Spacer-18 (hexaethyleneglycol))Cholesterol-3′ (SEQ ID NO: 4).